The introduction of West Nile virus (WNV) into North America has caused great concern due to its continued impact on human and veterinary health. Studies concerning the genetic changes occurring in the genome of WNV have been important in defining potential mechanisms of viral transmission and spread for both vertebrates and mosquitoes. Significantly, to date, there have been very limited studies on alterations of the phenotype of WNV in mosquitoes. Recent nucleic acid sequencing studies by the applicant's laboratory have described the microevolution of WNV since its introduction into the U.S. Subsequent sequence analysis of isolates collected in Texas during the 2003 WNV transmission season has revealed several genetically divergent isolates from 2003 that also differed phenotypically from earlier WNV isolates in plaque morphology, delayed production of CPE in Vero cell culture, and temperature sensitivity (ts) in comparison to isolates collected in Texas in 2002 and New York in 1999. Furthermore, isolates with a small plaque (sp) and /or ts phenotype exhibited reduced replication in cell culture, and mouse neuroinvasiveness studies also indicated that several of these isolates were attenuated in neuroinvasiveness. We hypothesize that these isolates will also be attenuated for mosquito infectivity. Elucidating the genetic and phenotypic changes in WNV over time will be critical to understanding the evolution, vector competence and epidemiology of WNV in North America and its impact on human and veterinary health. It is the hypothesis of this application that changes in the observed viral phenotypes in vertebrates and mosquitoes are conferred by nucleotide/amino acid substitutions in the genomes of these isolates. Thus, this application will sequence genome regions of isolates from 2006 to 2011 to identify nucleotide/amino acid substitutions. Following the identification of potential mutations, site-directed mutagenesis of an infectious clone based on the genome of the prototype WN-NY99, 382-99, will be undertaken in order to confirm the involvement of the specific genetic mutation. Identification of the nucleotides and deduced amino acid substitutions conferring viral phenotypes is essential to understanding the mechanisms of attenuation and virulence of WNV, and will be important to our understanding of how this virus will continue to influence human and veterinary health in the future. The following specific aims are proposed: 1. Compare nucleotide and deduced amino acid sequences of WNV isolate collected in North America during 2006-2011 to those collected between 1999 and 2005;2. Identification of phenotypic characteristics of WNV variant circulating in the U.S. during the 2006-2011 transmission seasons;3. Investigate the mechanism of attenuation of neuroinvasiveness of mouse attenuated WNV isolates;4. Correlate WNV phenotype in vertebrate cells with mosquito vector infection.